Human anti-peptidoglycan-IgG-mediated opsonophagocytosis is controlled by calcium mobilization in phorbol myristate acetate-treated U937 cells

Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca²⁺ release from intracellular Ca²⁺ stores and anti-PGN-IgGdependent Ca²⁺ mobilization is controlled via a phospholipase Cγ-2-mediated pathway. [BMB Reports 2015; 48(1): 36-41]     

Coregulation of collagenase and collagenase inhibitor production by phorbol myristate acetate in human skin fibroblasts

Phorbol myristate acetate (PMA), a tumor promotor known to stimulate collagenase production in fibroblasts and endothelial cells, was examined with regard to its ability to regulate the expression of the collagenase inhibitor secreted by human skin fibroblasts. Confluent human skin fibroblasts were incubated with concentrations of PMA ranging from 10(-11) to 10(-7) M, and the conditioned medium was analyzed by enzyme-linked immunosorbent assay for both immunoreactive collagenase and collagenase inhibitor. PMA stimulated the production of both collagenase and collagenase inhibitor in several cell lines to maximal rates that were very similar, 300 to 350 vs 230 to 330 pmol 10 micrograms DNA-1 48 h-1, respectively. Due to differences in the basal levels of expression of these proteins, such rates reflected a two- to sevenfold stimulation in collagenase production, in comparison to a more uniform two- to threefold enhancement in inhibitor synthesis. Production of inhibitor was 50% of maximal at 7 X 10(-9) M and maximal at 10(-7) M phorbol. This concentration-dependent effect was very similar to that observed for collagenase expression. Total protein synthesis by the phorbol-conditioned cells, as studied by incorporation of [3H]leucine into newly synthesized protein, was not significantly increased, nor was cellular DNA content. The onset of the effect of PMA on inhibitor production occurred between 4 and 8 h, was maximal by 8 h, and continued undiminished for at least another 64 h. After the first 8 h, inhibitor production continued at a roughly constant rate of approximately 10 pmol 10 micrograms DNA-1 h-1. Interestingly, following the removal of phorbol from culture medium, such fibroblasts continued to produce increased quantities of inhibitor protein for at least 72 h. Metabolic labeling studies in which fibroblasts were exposed to [3H]leucine followed by immunoprecipitation using inhibitor-specific antibody suggested that stimulation of inhibitor production by PMA was mediated via an increased synthesis of new inhibitor protein. Therefore, in response to the tumor promoter, PMA collagenase and collagenase inhibitor expression by human skin fibroblasts appear to be coregulated.     

Calcium ionophore and phorbol myristate acetate synergistically inhibited proteoglycan biosynthesis in articular chondrocytes by prostaglandin independent mechanism

In rabbit articular chondrocytes, phorbol myristate acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DG) and calcium ionophore (A23187), reduced the proteoglycan synthesis, in a dose-dependent manner. The combined treatment by PMA and A23187 resulted in an enhanced inhibition of proteoglycan production, indicating a synergistic effect. In presence of PMA or A23187, the release of prostaglandin E2 (PGE2) was dramatically increased. The addition of indomethacin and BW755c to chondrocytes stimulated by PMA or A23187, suppressed the liberation of PGE2, but did not stop the decrease of proteoglycan synthesis.     

Contrasting effects of phorbol dibutyrate and phorbol myristate acetate in rabbit aorta

In rat hepatocytes, active phorbol esters inhibited the alpha 1-adrenergic stimulation of phosphatidylinositol labeling with the expected potency order: phorbol myristate acetate (PMA) greater than phorbol dibutyrate (PDB). In contrast, in rabbit aorta the alpha 1-adrenergic action was inhibited dose-dependently by PDB but not by PMA. Similarly PDB (but not PMA) induced a strong contraction in rabbit aorta. The phorbol ester-induced contraction developed slowly, was dose-dependent and independent of extracellular calcium. These effects of PDB in rabbit aorta were neither inhibited by the protein kinase inhibitor H-7 nor mimicked by the synthetic diacylglycerol, OAG. Our results raise some doubts on the mechanism(s) through which the actions of PDB take place in rabbit aorta.     

The cartilage-resorbing protein catabolin is made by synovial fibroblasts and its production is increased by phorbol myristate acetate.

Pig synovial fibroblasts were shown to produce a protein that caused live cartilage to resorb its proteoglycan matrix in vitro. Fibroblasts were obtained either from synovial tissue digest or by allowing them to grow out of explants. The population derived from the digests was homogeneous and free of macrophage-like cells after two passages, but was still producing the cartilage-resorbing protein after seven passages. The active protein was found to have Mr 20,000 on gell filtration, and pI 4.8 on isoelectric focussing in polyacrylamide gel. It was indistinguishable from a protein with the same activity from pig mononuclear leucocytes, which has been called catabolin. Production of the protein was increased if the synovial fibroblasts were cultured with the tumour promoter phorbol 12-myristate 13-acetate. Fibroblasts from other sources (joint capsule and peritoneum) also apparently made the protein. The possibility that catabolin is the same as interleukin-1 is discussed: if they are, then the results suggest that fibroblasts can make an interleukin-1-life protein.     

Altered expression of integrins in RSV-transformed chick epiphyseal chondrocytes

Chondrocytes have been shown to express both in vivo and in vitro a number of integrins of the beta1-, beta3- and beta5-subfamilies (Biorheology 37 (2000) 109). Normal and v-Src-transformed chick epiphyseal chondrocytes (CEC) display different adhesion properties. While normal CEC with time in culture tends to increase their adhesion to the substrate by organizing focal adhesions and actin stress fibers, v-Src-transformed chondrocytes display a refractile morphology and disorganization of actin cytoskeleton. We wondered whether the reduced adhesion and spreading of v-Src-transformed chondrocytes could be ascribed to changes in integrin expression and/or function. Integrin expression by normal CEC is studied and compared to v-Src-transformed chick chondrocytes, using monoclonal and polyclonal antibodies to integrins alpha- and beta-chains. We show the presence of alpha1-, alpha3-, alphav-, alpha6-, beta1- and beta3-chains on CEC, with very low levels of alpha2- and alpha5-chains. Alphav chain associates with multiple beta subunits in normal and transformed chondrocytes. With the exception of alpha1- and alpha2-chains, the levels of the integrin chains analyzed are higher in transformed chondrocytes as compared with normal chondrocytes. In spite of the increased levels of integrin expression, transformed chondrocytes exhibit loss of focal adhesion and actin stress fibers and low adhesion activity on several extracellular matrix constituents. These observations raise the possibility that, in addition to its effects on global pattern of integrin expression, v-Src can influence integrin function in chondrocytes.     

Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in Rb-deficient fibroblasts.

Fibroblasts prepared from retinoblastoma (Rb) gene-negative mouse embryos exhibit a shorter G1 phase of the growth cycle and smaller size than wild-type cells. In addition, the mutant cells are no longer inhibited by low levels of cycloheximide at any point in G1 but do remain sensitive to serum withdrawal until late in G1. Certain cell cycle-regulated genes showed no temporal or quantitative differences in expression. In contrast, cyclin E expression in Rb-deficient cells is deregulated in two ways. Cyclin E mRNA is generally derepressed in mutant cells and reaches peak levels about 6 h earlier in G1 than in wild-type cells. Moreover, cyclin E protein levels are higher in the Rb-/- cells than would be predicted from the levels of its mRNA. Thus, the selective growth advantage conferred by Rb gene deletion during tumorigenesis may be explained in part by changes in the regulation of cyclin E. In addition, the mechanisms defining the restriction point of late G1 may consist of at least two molecular events, one cycloheximide sensitive and pRb dependent and the other serum sensitive and pRb independent.     

Imaging native beta-actin mRNA in motile fibroblasts

Nuclease-resistant, cytoplasmically resident molecular beacons were used to specifically label beta-actin mRNA in living and motile chicken embryonic fibroblasts. beta-actin mRNA signals were most abundant in active lamellipodia, which are protrusions that cells extend to adhere to surfaces. Time-lapse images show that the immediate sources of beta-actin mRNA for nascent lamellipodia are adjacent older protrusions. During the development of this method, we observed that conventional molecular beacons are rapidly sequestered in cell nuclei, leaving little time for them to find and bind to their cytoplasmic mRNA targets. By linking molecular beacons to a protein that tends to stay within the cytoplasm, nuclear sequestration was prevented, enabling cytoplasmic mRNAs to be detected and imaged. Probing beta-actin mRNA with these cytoplasmically resident molecular beacons did not affect the motility of the fibroblasts. Furthermore, mRNAs bound to these probes undergo translation within the cell. The use of cytoplasmically resident molecular beacons will enable further studies of the mechanism of beta-actin mRNA localization, and will be useful for understanding the dynamics of mRNA distribution in other living cells.     

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.     

Vasoactive intestinal peptide enhances phorbol myristate acetate-induced chemiluminescence in human lymphocytes.

Phorbol-myristate-acetate (PMA) induced in lymphocytes the production or reactive oxygen intermediates in a process which was stimulated by the presence of vasoactive intestinal peptide (VIP) in a dose-dependent response at VIP concentrations in the range 10(-11)-10(-8) M. The dissociation constant for the high-affinity receptors of VIP agreed with the ID50 of the activation of adenylate cyclase, and the ID50 for the stimulation by VIP of PMA-induced chemiluminescence, which were close to 0.2 nM VIP. Forskolin produced in lymphocytes an effect quite similar to VIP. A comparison of the response to VIP and forskolin of lymphocytes and monocytes showed that, in contrast to forskolin, VIP failed to induce the above described effect in monocytes. A possible mechanism involving protein kinase C, which is activated by PMA, and an intracellular signal linked to VIP receptors is pointed out. This study further supports a role for VIP as a mediator in the neuroimmune system.     

Functional analysis of elements affecting expression of the beta-actin gene of carp.

Regulatory regions of the beta-actin gene of the common carp (Cyprinus carpio) have been examined by linking upstream, 5'-flanking sequences and regions of the first intron to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. By analysis of the mRNA products and encoded CAT activity, we have identified four putative regions that influence expression: (i) a negative regulatory region 2,300 to 1,100 base pairs (bp) ahead of the gene; (ii) a proximal promoter element, containing the highly conserved CCAAT, CC(A/T)6GG, and TATA boxes, that is within the first 204 bp upstream of the initiation site; (iii) a negative element of 426 bp in the 5' region of the first intron; and (iv) a positive 304-bp element near the end of the first intron that contains highly conserved sequences found in all characterized beta-actin genes. The positive intron element is not a classical enhancer; it is position and orientation dependent, as has been observed in other housekeeping genes in vertebrates. Depending on the elements joined together, CAT gene expression can be modulated more than 500-fold in transfected mouse cells.     

Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate

A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.